Chemical compounds

ABSTRACT

This invention relates to polymorphisms in the human SLC10A2 gene and corresponding novel allelic polypeptides encoded thereby. The invention also relates to methods and materials for analysing allelic variation in the SLC10A2 gene, and to the use of SLC10A2 polymorphism in treatment of diseases with SLC10A2 drugs.

[0001] This invention relates to polymorphisms in the human ilealsodium-dependent bile acid transporter gene (SLC10A2) and correspondingnovel allelic polypeptides encoded thereby. The invention also relatesto methods and materials for analysing allelic variation in the SLC10A2gene, and to the use of SLC10A2 polymorphism in treatment of diseaseswith SLC10A2 drugs.

[0002] Bile acids are synthesised in the liver from cholesterol andsecreted into the small intestine, where they are used for theabsorption of fat, fat-soluble vitamins, and cholesterol. These biteacids are reabsorbed from the small intestine and resecreted into bilefrom the liver via the venous portal circulation. This process isextremely efficient with less than 10% of bile acids being excreted viafaeces.

[0003] The active uptake of bile acids from the small intestine ismediated by a Na⁺-dependent transport system located at the apicalbrush-border membrane of the ileocyte. In 1995 Wong et al isolated. thehuman ileal Na⁺/bile acid cotransporter cDNA (gene SLC10A2) and mappedit to chromosome 13q33. Whilst cloning and characterising the gene adysfunctional missense mutation was identified in a patient diagnosedwith Crohn's disease. This mutation involved a single C to T transition,resulting in a proline to serine substitution at amino acid position290. Oelkers et al found that the mutation did not interfere withprotein expression or trafficking to the cell surface, but blocked thetransport activity of taurocholate and other bile acids. Inheritance ofthis mutation was confirmed by single-strand confirmation polymorphismanalysis and DNA sequencing.

[0004] In 1997 Oelkers et al reported that inherited loss-of-functionmutations in SLC10A2 can cause primary bile acid malabsorption (PBAM).Studies of PBAM patients suggested autosomal recessive inheritance, andthis was supported by the lack of clinical symptoms in the proband'sson. PBAM is involved in a lowered LDL cholesterol level as increasedloss of bile acids lowers plasma LDL cholesterol levels due to thediversion of hepatic cholesterol to bile acid synthesis. The probanddescribed in the Oelkers et al paper had a LDL cholesterol levelconsistently below the 10^(th) percentile. This has been used as asurrogate marker for ileal Na⁺/bile acid co-transporter function.

[0005] It is well-known that hyperlipidaemic conditions associated withelevated concentrations of total cholesterol and low-density lipoproteincholesterol are major risk factors for cardiovascular atheroscleroticdisease (for instance “Coronary Heart Disease: Reducing the Risk; aWorldwide View” Assman G., Carmena R. Cullen P. et al; Circulation 1999,100, 1930-1938 and “Diabetes and Cardiovascular Disease: A Statement forHealthcare Professionals from the American Heart Association” Grundy S,Benjamin I., Burke G., et al; Circulation, 1999, 100, 1134-46).Interfering with the circulation of bile acids within the lumen of theintestinal tracts is found to reduce the level of cholesterol. Previousestablished therapies to reduce the concentration of cholesterolinvolve, for instance, treatment with HMG-CoA reductase inhibitors,preferably statins such as simvastatin and fluvastatin, or treatmentwith bile acid binders, such as resins. Frequently used bile acidbinders are for instance cholestyramine and cholestipol. One recentlyproposed therapy (“Bile Acids and Lipoprotein Metabolism: a Renaissancefor Bile Acids in the Post Statin Era” Angelin B, Eriksson M, Rudling M;Current Opinion on Lipidology, 1999, 10, 269-74) involved the treatmentwith substances with an ileal bile acid transport (IBAT) inhibitoryeffect.

[0006] Re-absorption of bile acid from the gastrointestinal tract is anormal physiological process which mainly takes place in the ileum bythe IBAT mechanism. Inhibitors of IBAT can be used in the treatment ofhypercholesterolaemia (see for instance “Interaction of bile acids andcholesterol with nonsystemic agents having hypocholesterolaemicproperties”, Biochemica et Biophysica Acta, 1210 (1994) 255-287). Thus,suitable compounds having such inhibitory IBAT activity are also usefulin the treatment of hyperlipidaemic conditions. Substitutedbenzothiazepines possessing such IBAT inhibitory activity have beendescribed, see for instance hypolipidaemic benzothiazepine compoundsdescribed in WO 93/16055, WO 94/18183, WO 94/18184, WO 96/05188, WO96/08484, WO 96/16051, WO 97/33882, WO 98/38182, WO 99/35135, WO98/40375 and EP 0 864 582.

[0007] Thus SLC10A2 is a target for cholesterol lowering drugs. Geneticpolymorphism in the gene may lead to variation in response to such drugsin the population.

[0008] A common polymorphism A171S was published, in 1997 by Oelkers etal. This polymorphism, is found in exon 3, and is present in 28% ofindividuals in a Caucasian control population of 104 unaffectedindividuals. It encodes a G to T transversion which results in analanine to serine substitution in the transporter's third predictedtransmembrane domain. Oelkers et al report that this polymorphism doesnot affect ileal Na⁺/bile acid cotransporter protein expression ortaurocholate uptake. This invention relates to further geneticpolymorphisms in SLC10A2.

REFERENCES

[0009] Wong, M. H., Oelkers, P., and P. A Dawson. 1995. Identificationof a mutation in the ileal sodium-dependent bile acid transporter genethat abolishes transport activity. J. Biol. Chem. 270,27228-27234.

[0010] Wong, M. H., Nagesh Rao, P., Pettenati, M. J., and P. A. Dawson.1996. Localisation of the ileal sodium-bile acid cotransporter gene(SLC10A2) to human chromosome 13q33. Genomics 33, 538-540.

[0011] Oelkers, P., Kirby, L. C., Heubi, J. E., and P. A. Dawson. 1997.Primary bile acid malabsorption caused by mutations in the ilealsodium-dependent bile acid transporter gene (SLC10A2). J. Clin. Invest.99, 1880-1887.

[0012] The present invention is based on the discovery of polymorphismsin SLC10A2. In particular, we have found 16 polynucleotide polymorphismsin the SLC10A2 gene, 6 of which lead to changes in the sequence ofexpressed protein.

[0013] According to one aspect of the present invention there isprovided a method for the diagnosis of a polymorphism in SLC10A2 in ahuman, which method comprises determining the sequence of the human atat least one polymorphic position and determining the status of thehuman by reference to polymorphism in SLC10A2. Preferred polymorphicpositions are one or more of the following positions:

[0014] positions582,664,727,792,890,1073,1103,1384,1466,1484,1545,1646,1683 and 1765 asdefined by the position in SEQ ID NO: 1;

[0015] position 1982 as defined by the position in SEQ ID NO: 2;

[0016] position 258 as defined by the position in SEQ ID NO: 3; and

[0017] positions 65, 98, 159, 290, 296 and 316 as defined by theposition in SEQ ID NO: 4.

[0018] The term human includes both a human having or suspected ofhaving a SLC10A2 mediated disease and an asymptomatic human who may betested for predisposition or susceptibility to such disease. At eachposition the human may be homozygous for an allele or the human may be aheterozygote.

[0019] The term polymorphism includes single nucleotide substitution,nucleotide insertion and nucleotide deletion which in the case ofinsertion and deletion includes insertion or deletion of one or morenucleotides at a position of a gene and corresponding alterations inexpressed protein.

[0020] In one embodiment of the invention preferably the method fordiagnosis described herein is one in which the polymorphism in SLC10A2is any one of the following: Allele in SEQ ID Variant Ref. SequencePosition NO x Allele SEQ ID NO: 1 582 C G SEQ ID NO: 1 664 T C SEQ IDNO: 1 727 C T SEQ ID NO: 1 792 C T SEQ ID NO: 1 890 G A SEQ ID NO: 11073 G A SEQ ID NO: 1 1103 C T SEQ ID NO: 1 1384 G T SEQ ID NO: 1 1466 CT SEQ ID NO: 1 1484 T C SEQ ID NO: 1 1545 G A SEQ ID NO: 1 1646 A T SEQID NO: 1 1683 T C SEQ ID NO: 1 1765 T C SEQ ID NO: 2 1982 T C SEQ ID NO:3 258 G A SEQ ID NO: 4 65 P L SEQ ID NO: 4 98 V I SEQ ID NO: 4 159 V ISEQ ID NO: 4 290 P S SEQ ID NO: 4 296 F L SEQ ID NO: 4 316 G E

[0021] The method for diagnosis is preferably one in which thepolynucleotide sequence is determined by a method selected fromamplification refractory mutation system, restriction fragment lengthpolymorphism and primer extension.

[0022] The status of the individual may be determined by reference toallelic variation at any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18 or more positions.

[0023] The test sample of nucleic acid is conveniently a sample ofblood, bronchoalveolar lavage fluid, sputum, or other body fluid ortissue obtained from an individual. It will be appreciated that the testsample may equally be a nucleic acid sequence corresponding to thesequence in the test sample, that is to say that all or a part of theregion in the sample nucleic acid may firstly be amplified using anyconvenient technique e.g. PCR, before analysis of allelic variation.

[0024] It will be apparent to the person skilled in the art that thereare a large number of analytical procedures which may be used to detectthe presence or absence of variant nucleotides at one or morepolymorphic positions of the invention. In general, the detection ofallelic variation requires a mutation discrimination technique,optionally an amplification reaction and optionally a signal generationsystem. Table 1 lists a number of mutation detection techniques, somebased on the PCR. These may be used in combination with a number ofsignal generation systems, a selection of which is listed in Table 2.Further amplification techniques are listed in Table 3. Many currentmethods for the detection of allelic variation are reviewed by Nollau etal., Clin. Chem. 43, 1114-1120, 1997; and in standard textbooks, forexample “Laboratory Protocols for Mutation Detection”, Ed. by U.Landegren, Oxford University Press, 1996 and “PCR”, 2^(nd) Edition byNewton & Graham, BIOS Scientific Publishers Limited, 1997.

[0025] Abbreviations: TABLE 1 Mutation Detection Techniques ALEX ™Amplification refractory mutation system linear extension APEX Arrayedprimer extension ARMS ™ Amplification refractory mutation system b-DNABranched DNA bp base pair CMC Chemical mismatch cleavage COPSCompetitive oligonucleotide priming system DGGE Denaturing gradient gelelectrophoresis ELISA Enzyme Linked ImmunoSorbent Assay FRETFluorescence resonance energy transfer LCR Ligase chain reaction MASDAMultiple allele specific diagnostic assay NASBA Nucleic acid sequencebased amplification OLA Oligonucleotide ligation assay PCR Polymerasechain reaction PTT Protein truncation test RFLP Restriction fragmentlength polymorphism SDA Strand displacement amplification SNP Singlenucleotide polymorphism SSCP Single-strand conformation polymorphismanalysis SSR Self sustained replication TGGE Temperature gradient gelelectrophoresis

[0026] General: DNA sequencing, Sequencing by hybridisation

[0027] Scanning: PTT*, SSCP, DGGE, TGGE, Cleavase, Heteroduplexanalysis, CMC, Enzymatic mismatch cleavage

[0028] *Note: not useful for detection of promoter polymorphisms.

[0029] Hybridisation Based

[0030] Solid phase hybridisation: Dot blots, MASDA, Reverse dot blots,Oligonucleotide arrays (DNA Chips).

[0031] Solution phase hybridisation: Taqman™—U.S. Pat. No. 5,210,015 &U.S. Pat. No. 5,487,972 (Hoffmann-La Roche), Molecular Beacons—Tyagi etal (1996), Nature Biotechnology, 14, 303; WO 95/13399 (Public HealthInst., New York)

[0032] Extension Based: ARMS™, ALEX™—European Patent No. EP 332435 B 1(Zeneca Limited), COPS—Gibbs et al (1989), Nucleic Acids Research, 17,2347.

[0033] Incorporation Based: Mini-sequencing, APEX

[0034] Restriction Enzyme Based: RFLP, Restriction site generating PCR

[0035] Ligation Based: OLA

[0036] Other: Invader assay

[0037] Table 2—Signal Generation or Detection Systems

[0038] Fluorescence: FRET, Fluorescence quenching, Fluorescencepolarisation—United Kingdom Patent No. 2228998 (Zeneca Limited)

[0039] Other: Chemiluminescence, Electrochemiluminescence, Raman,Radioactivity, Colorimetric, Hybridisation protection assay, Massspectrometry

[0040] Table 3—Further Amplification Methods

[0041] SSR, NASBA, LCR, SDA, b-DNA

[0042] Table 4—Protein Variation Detection Methods

[0043] Immunoassay

[0044] Immunohistology

[0045] Peptide sequencing

[0046] Preferred mutation detection techniques include ARMS™, ALEX™,COPS, Taqman, Molecular Beacons, RFLP, and restriction site based PCRand FRET techniques. Immunoassay techniques are known in the art e.g. APractical Guide to ELISA by D M Kemeny, Pergamon Press 1991; Principlesand Practice of Immunoassay, 2^(nd) edition, C P Price & D J Newman,1997, published by Stockton Press in USA & Canada and by MacmillanReference in the United Kingdom.

[0047] Particularly preferred methods include ARMS™ and RFLP basedmethods. ARMS™ is an especially preferred method.

[0048] In a further aspect, the diagnostic methods of the invention areused to assess the pharmacogenetics of a SLC10A2 drug.

[0049] Assays, for example reporter-based assays, may be devised todetect whether one or more of the above polymorphisms affecttranscription levels and/or message stability.

[0050] Individuals who carry particular allelic variants of the SLC10A2gene may therefore exhibit differences in their ability to regulateprotein biosynthesis under different physiological conditions and willdisplay altered abilities to react to different diseases. In addition,differences arising as a result of allelic variation may have a directeffect on the response of an individual to drug therapy. The diagnosticmethods of the invention may be useful both to predict the clinicalresponse to such agents and to determine therapeutic dose.

[0051] In a further aspect, the diagnostic methods of the invention, areused to assess the predisposition and/or susceptibility of an individualto diseases mediated by SLC10A2. This may be particularly relevant inthe development of hyperlipoproteinemia and cardiovascular disease andthe present invention may be used to recognise individuals who areparticularly at risk from developing these conditions.

[0052] In a further aspect, the diagnostic methods of the invention areused in the development of new drug therapies which selectively targetone or more allelic variants of the SLC10A2 gene. Identification of alink between a particular allelic variant and predisposition to diseasedevelopment or response to drug therapy may have a significant impact onthe design of new drugs. Drugs may be designed to regulate thebiological activity of variants implicated in the disease process whilstminimising effects on other variants.

[0053] In a further diagnostic aspect of the invention the presence orabsence of variant nucleotides is detected by reference to the loss orgain of, optionally engineered, sites recognised by restriction enzymes.

[0054] According to another aspect of the present invention there isprovided a human SLC10A2 gene or its complementary strand comprising avariant allelic polymorphism at one or more of positions defined hereinor a fragment thereof of at least 20 bases comprising at least one novelpolymorphism.

[0055] Fragments are at least 17 bases, more preferably at least 20bases, more preferably at least 30 bases.

[0056] According to another aspect of the present invention there isprovided a polynucleotide comprising at least 20 bases of the humanSLC10A2 gene and comprising an allelic variant selected from any one ofthe following: Variant Ref. Sequence Position Allele SEQ ID NO: 1 582 GSEQ ID NO: 1 664 C SEQ ID NO: 1 727 T SEQ ID NO: 1 792 T SEQ ID NO: 1890 A SEQ ID NO: 1 1073 A SEQ ID NO: 1 1103 T SEQ ID NO: 1 1384 T SEQ IDNO: 1 1466 T SEQ ID NO: 1 1484 C SEQ ID NO: 1 1545 A SEQ ID NO: 1 1646 TSEQ ID NO: 1 1683 C SEQ ID NO: 1 1765 C SEQ ID NO: 2 1982 C SEQ ID NO: 3258 A

[0057] According to another aspect of the present invention there isprovided a human SLC10A2 gene or its complementary strand comprising apolymorphism, preferably corresponding with one or more the positionsdefined herein or a fragment thereof of at least 20 bases comprising atleast one polymorphism.

[0058] Fragments are at least 17 bases, more preferably at least 20bases, more preferably at least 30 bases.

[0059] The invention further provides a nucleotide primer which candetect a polymorphism of the invention.

[0060] According to another aspect of the present invention there isprovided an allele specific primer capable of detecting a SLC10A2 genepolymorphism, preferably at one or more of the positions as definedherein.

[0061] An allele specific primer is used, generally together with aconstant primer, in an amplification reaction such as a PCR reaction,which provides the discrimination between alleles through selectiveamplification of one allele at a particular sequence position e.g. asused for ARMS™ assays. The allele specific primer is preferably 17-50nucleotides, more preferably about 17-35 nucleotides, more preferablyabout 17-30 nucleotides.

[0062] An allele specific primer preferably corresponds exactly with theallele to be detected but derivatives thereof are also contemplatedwherein about 6-8 of the nucleotides at the 3′ terminus correspond withthe allele to be detected and wherein up to 10, such as up to 8, 6, 4,2, or 1 of the remaining nucleotides may be varied without significantlyaffecting the properties of the primer.

[0063] Primers may be manufactured using any convenient method ofsynthesis. Examples of such methods may be found in standard textbooks,for example “Protocols for Oligonucleotides and Analogues; Synthesis andProperties,” Methods in Molecular Biology Series; Volume 20; Ed. SudhirAgrawal, Humana ISBN: 0-89603-247-7; 1993; 1^(st) Edition. If requiredthe primer(s) may be labelled to facilitate detection.

[0064] According to another aspect of the present invention there isprovided an allele-specific oligonucleotide probe capable of detecting aSLC10A2 gene polymorphism, preferably at one or more of the positionsdefined herein.

[0065] The allele-specific oligonucleotide probe is preferably 17-50nucleotides, more preferably about 17-35 nucleotides, more preferablyabout 17-30 nucleotides.

[0066] The design of such probes will be apparent to the molecularbiologist of ordinary skill. Such probes are of any convenient lengthsuch as up to 50 bases, up to 40 bases, more conveniently up to 30 basesin length, such as for example 8-25 or 8-15 bases in length. In generalsuch probes will comprise base sequences entirely complementary to thecorresponding wild type or variant locus in the gene. However, ifrequired one or more mismatches may be introduced, provided that thediscriminatory power of the oligonucleotide probe is not undulyaffected. The probes of the invention may carry one or more labels tofacilitate detection.

[0067] According to another aspect of the present invention there isprovided an allele specific primer or an allele specific oligonucleotideprobe capable of detecting a SLC10A2 gene polymorphism at one of thepositions defined herein.

[0068] According to another aspect of the present invention there isprovided a diagnostic kit comprising an allele specific oligonucleotideprobe of the invention and/or an allele-specific primer of theinvention.

[0069] The diagnostic kits may comprise appropriate packaging andinstructions for use in the methods of the invention. Such kits mayfurther comprise appropriate buffer(s) and polymerase(s) such asthermostable polymerases, for example taq polymerase.

[0070] In another aspect of the invention, the single nucleotidepolymorphisms of this invention may be used as genetic markers inlinkage studies. This particularly applies to the polymorphisms at 1765in SEQ ID NO 1 and at 258 in SEQ ID NO 3 because of their relativelyhigh frequencies (see below). The SLC10A2 gene has been mapped to humanchromosome 13q33 (Wong et al, Genomics 33: 538-540, 1996).

[0071] Low frequency polymorphisms may be particularly useful forhaplotyping as described below. A haplotype is a set of alleles found atlinked polymorphic sites (such as within a gene) on a single (paternalor maternal) chromosome. If recombination within the gene is random,there may be as many as 2^(n) haplotypes, where 2 is the number ofalleles at each SNP and n is the number of SNPs. One approach toidentifying mutations or polymorphisms which are correlated withclinical response is to carry out an association study using all thehaplotypes that can be identified in the population of interest. Thefrequency of each haplotype is limited by the frequency of its rarestallele, so that SNPs with low frequency alleles are particularly usefulas markers of low frequency haplotypes. As particular mutations orpolymorphisms associated with certain clinical features, such as adverseor abnormal events, are likely to be of low frequency within thepopulation, low frequency SNPs may be particularly useful in identifyingthese mutations (for examples see: Linkage disequilibrium at thecystathionine beta synthase (CBS) locus and the association betweengenetic variation at the CBS locus and plasma levels of homocysteine.Ann Hum Genet (1998) 62:481-90, De Stefano V, Dekou V, Nicaud V, ChasseJF, London J, Stansbie D, Humphries S E, and Gudnason V; and Variationat the von willebrand factor (vWF) gene locus is associated with plasmavWF:Ag levels: identification of three novel single nucleotidepolymorphisms in the vWP gene promoter. Blood (1999) 93:4277-83,Keightley A M, Lam Y M, Brady J N, Cameron C L, Lillicrap D).

[0072] According to another aspect of the present invention there isprovided a computer readable medium comprising at least one novelsequence of the invention stored on the medium. The computer readablemedium may be used, for example, in homology searching, mapping,haplotyping, genotyping or pharmacogenetic analysis.

[0073] According to another aspect of the present invention there isprovided a method of treating a human in need of treatment with aSLC10A2 drug in which the method comprises:

[0074] i) diagnosis of a polymorphism in SLC10A2 in the human, whichdiagnosis preferably comprises determining the sequence at one or moreof the following positions:

[0075] positions582,664,727,792,890,1073,1103,1384,1466,1484,1545,1646,1683 and 1765 asdefined by the position in SEQ ID NO: 1;

[0076] position 1982 as defined by the position in SEQ ID NO: 2;

[0077] position 258 as defined by the position in SEQ ID NO: 3; and

[0078] positions 65, 98, 159, 290, 296 and 316 as defined by theposition in SEQ ID NO: 4.

[0079] and determining the status of the human by reference topolymorphism in the SLC10A2 gene; and

[0080] ii) administering an effective amount of the drug.

[0081] Preferably determination of the status of the human is clinicallyuseful. Examples of clinical usefulness include deciding which drug ordrugs to administer and/or in deciding on the effective amount of thedrug or drugs. The term “SLC10A2 drug” means either that interactionwith SLC10A2 in humans is an aspect of a drug exerting its pharmceuticaleffect in man or that SCL10A2 is involved in the biological pathwaythrough which drug exerts its pharmceutical effect in man or that thedrug is transported by SLC10A2. For example, substitutedbenzothiazepines possessing SLC10A2 inhibitory activity have beendescribed, see for instance hypolipidaemic benzothiazepine compoundsdescribed in WO 93/16055, WO 94/18183, WO 94/18184, WO 96/05188, WO96/08484, WO 96/16051, WO 97/33882, WO 98/38182, WO 99/35135, WO98/40375 and EP 0 864 582.

[0082] According to another aspect of the present invention there isprovided an allelic variant of human SLC10A2 polypeptide comprising atleast one of the following: Position Allelic variant SEQ ID NO: 4 65 LSEQ ID NO: 4 98 I SEQ ID NO: 4 159 I SEQ ID NO: 4 290 S SEQ ID NO: 4 296L SEQ ID NO: 4 316 E

[0083] or a fragment thereof comprising at least 10 amino acids providedthat the fragment comprises at least one allelic variant.

[0084] Fragments of polypeptide are at least 10 amino acids, morepreferably at least 15 amino acids, more preferably at least 20 aminoacids.

[0085] According to another aspect of the present invention there isprovided an antibody specific for an allelic variant of human SLC10A2polypeptide as described herein.

[0086] Antibodies can be prepared using any suitable method. Forexample, purified polypeptide may be utilized to prepare specificantibodies. The term “antibodies” is meant to include polycionalantibodies, monoclonal antibodies, and the various types of antibodyconstructs such as for example F(ab′)₂, Fab and single chain Fv.Antibodies are defined to be specifically binding if they bind theallelic variant of SLC10A2 with a K_(a) of greater than or equal toabout 10⁷ M⁻¹. Affinity of binding can be determined using conventionaltechniques, for example those described by Scatchard et al., Ann. N.Y.Acad. Sci., 51:660 (1949).

[0087] Polyclonal antibodies can be readily generated from a variety ofsources, for example, horses, cows, goats, sheep, dogs, chickens,rabbits, mice or rats, using procedures that are well-known in the art.In general, antigen is administered to the host animal typically throughparenteral injection. The immunogenicity of antigen may be enhancedthrough the use of an adjuvant, for example, Freund's complete orincomplete adjuvant. Following booster immunizations, small samples ofserum are collected and tested for reactivity to antigen. Examples ofvarious assays useful for such determination include those described in:Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988; as well as procedures such ascountercurrent immuno-electrophoresis (CIEP), radioimmunoassay,radioimmunoprecipitation, enzyme-linked immuno-sorbent assays (ELISA),dot blot assays, and sandwich assays, see U.S. Pat. Nos. 4,376,110 and4,486,530.

[0088] Monoclonal antibodies may be readily prepared using well-knownprocedures, see for example, the procedures described in U.S. Pat. Nos.RE 32,011, 4,902,614, 4,543,439 and 4,411,993; Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Plenum Press,Kennett, McKearn, and Bechtol (eds.), (1980).

[0089] The monoclonal antibodies of the invention can be produced usingalternative techniques, such as those described by Alting-Mees et al.,“Monoclonal Antibody Expression Libraries: A Rapid Alternative toHybridomas”, Strategies in Molecular Biology 3: 1-9 (1990) which isincorporated herein by reference. Similarly, binding partners can beconstructed using recombinant DNA techniques to incorporate the variableregions of a gene that encodes a specific binding antibody. Such atechnique is described in Larrick et al., Biotechnology, 7: 394 (1989).

[0090] Once isolated and purified, the antibodies may be used to detectthe presence of antigen in a sample using established assay protocols,see for example “A Practical Guide to ELISA” by D. M. Kemeny, PergamonPress, Oxford, England.

[0091] According to another aspect of the invention there is provided adiagnostic kit comprising an antibody of the invention.

[0092] The invention will now be illustrated but not limited byreference to the following Examples. All temperatures are in degreesCelsius.

[0093] In the Examples below, unless otherwise stated, the followingmethodology and materials have been applied.

[0094] AMPLITAQ™ available from Perkin-Elmer Cetus, is used as thesource of thermostable DNA polymerase.

[0095] General molecular biology procedures can be followed from any ofthe methods described in “Molecular Cloning—A Laboratory Manual” SecondEdition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory,1989) or in “Current Protocols in Molecular Biology Volumes 1-3, editedby F M Asubel, R Brent and R E Kingston; published by John Wiley, 1998.

[0096] Electropherograms were obtained in a standard manner: data wascollected by ABI377 data collection software and the wave form generatedby ABI Prism sequencing analysis (2.1.2).

EXAMPLE 1

[0097] Identification of Polymorphisms

[0098] 1. Methods

[0099] DNA Preparation

[0100] DNA was prepared from frozen blood samples collected in EDTAfollowing protocol I (Molecular Cloning: A Laboratory Manual, p392,Sambrook, Fritsch and Maniatis, 2^(nd) Edition, Cold Spring HarborPress, 1989) with the following modifications. The thawed blood wasdiluted in an equal volume of standard saline citrate instead ofphosphate buffered saline to remove lysed red blood cells. Samples wereextracted with phenol, then phenol/chloroform and then chloroform ratherthan with three phenol extractions. The DNA was dissolved in deionisedwater.

[0101] Template Preparation

[0102] Templates were prepared by PCR using the oligonucleotide primersand annealing temperatures set out below. The extension temperature was72° and denaturation temperature 94°. Generally 50 ng of genomic DNA wasused in each reaction and subjected to 35 cycles of PCR. Where describedbelow, the primary fragment was diluted {fraction (1/100)} and twomicrolitres were used as template for amplification of secondaryfragments. PCR was performed in two stages (primary fragment thensecondary fragment) to ensure specific amplification of the desiredtarget sequence.

[0103] 2 Polymorphisms in SLC10A2

[0104] The following polymorphisms were discovered in SLC10A2 using bothcDNA and genomic DNA templates. Published Novel Ref. Sequence PositionAllele Allele Frequency Comment SEQ ID NO: 1 582 C G 1/56 5′UTR SEQ IDNO: 1 664 T C 1/56 silent SEQ ID NO: 1 727 C T 1/56 silent SEQ ID NO: 1792 C T 1/46 P65L SEQ ID NO: 4 SEQ ID NO: 1 890 G A 3/58 V98I SEQ ID NO:4 SEQ ID NO: 1 1073 G A 1/56 V159I SEQ ID NO: 4 SEQ ID NO: 1 1103 C T1/56 silent SEQ ID NO: 1 1384 G T 1/56 silent SEQ ID NO: 1 1466 C T 1/56P290S SEQ ID NO: 4 SEQ ID NO: 1 1484 T C 1/56 F296L SEQ ID NO: 4 SEQ IDNO: 1 1545 G A 1/56 G316E SEQ ID NO: 4 SEQ ID NO: 1 1646 A T 1/56 3′UTRSEQ ID NO: 1 1683 T C 1/56 3′UTR SEQ ID NO: 1 1765 T C 30/50  3′UTR SEQID NO: 2 1982 T C 1/56 intron 1 SEQ ID NO: 3 258 G A 16/56  intron 5

[0105]

1 4 1 3779 DNA Homo sapiens 1 ttctattgaa agggaaatgg gagaacaatatgtgttccta tggctcagtc cctataagat 60 tctgtactat tcagagttga ttttaagtgtcacttaactg aaattatcca acaaaccttc 120 atggcatgaa acattaacac agctctttttatatggcatg gttcctatgg ctcaatccct 180 ataagattct gtactagttc agagttgattttaaaagtca cttaactgaa attatccaac 240 aaaccctcga ggacattaaa cattaacgtggctcttttta tatggcatgg ttcattatca 300 tgccaataaa tgattaatcg taactctctgtcttgaccaa taattttgct ggacttttgt 360 gattcacaac gtgctctgtg ttgtaatgctacctcttgaa actgacatcc tagctttatt 420 gttttttatt acttccctaa ggtggctttcaaaagagaca ccaagtgaca tatttttagg 480 aggggtttaa aagtttgatg gggtagaagtaaacgttgct taactcaacc agcagcagag 540 ccagggccca gggaccagcg cttctgtggacttggccttt ccagcagcag acccagcaat 600 gaatgatccg aacagctgtg tggacaatgcaacagtttgc tctggtgcat cctgtgtggt 660 acctgagagc aatttcaata acatcctaagtgtggtccta agtacggtgc tgaccatcct 720 gttggccttg gtgatgttct ccatgggatgcaacgtggaa atcaagaaat ttctagggca 780 cataaagcgg ccgtggggca tttgtgttggcttcctctgt cagtttggaa tcatgcccct 840 cacaggattc atcctgtcgg tggcctttgacatcctcccg ctccaggccg tagtggtgct 900 cattatagga tgctgccctg gaggaactgcctccaatatc ttggcctatt gggtcgatgg 960 cgacatggac ctgagcgtca gcatgaccacatgctccaca ctgcttgccc tcggaatgat 1020 gccgctgtgc ctccttatct ataccaaaatgtgggtcgac tctgggagca tcgtaattcc 1080 ctatgataac ataggtacat ctctggttgctctcgttgtt cctgtttcca ttggaatgtt 1140 tgttaatcac aaatggcccc aaaaagcaaagatcatactt aaaattgggt ccatcgcggg 1200 cgccatcctc attgtgctca tagctgtggttggaggaata ttgtaccaaa gcgcctggat 1260 cattgctccc aaactgtgga ttataggaacaatatttcct gtggcgggtt actccctggg 1320 gtttcttctg gctagaattg ctggtctaccctggtacagg tgccgaacgg ttgcttttga 1380 aacggggatg cagaacacgc agctatgttccaccatcgtt cagctctcct tcactcctga 1440 ggagctcaat gtcgtattca ccttcccgctcatctacagc attttccagc tcgcctttgc 1500 cgcaatattc ttaggatttt atgtggcatacaagaaatgt catggaaaaa acaaggcaga 1560 aattccagag agcaaagaaa atggaacggagccagagtca tcgttttata aggcaaatgg 1620 aggatttcaa cctgacgaaa agtagacatcaagtggacaa aacagacgag ttccaaatta 1680 cgttcttaaa ccgtaactat atttaattatttgttttggt aggacagttg gcagaaaaga 1740 gttaaagtga aaattggaat ttcattggaattcatgtatt ggtttcagta ccaagtgact 1800 ggtggcccaa ttctttaatg ggacaaatattgtttcctat atatatgtat atgttttata 1860 tatgtatgta tactcatata gatatattgtcattgaaata ttcccccaaa atattctcag 1920 actaaacctg acatagggaa caccgagaatgaaaacatcg ttaacaccaa aactgaattc 1980 ttatgcagaa tttcctagcc catagatgacaacctgagtt tctgtatgtt aaagtagatg 2040 taatgaatta ttattattac agtggtcacgattttcttca gtgtttatga ttataaaaat 2100 tgacatgaac atctttcact gacattttaatcattatttt aaaagctttg caacctatat 2160 atttatataa ctttgtaata taacatgggcaaatatctga cttcagtatt tttaaaaagt 2220 tgccttctcc agtggcagtc caaaagcagaaatgagagga aattattaca aaatagaatt 2280 caataaccat attggatgca ggctcttaactcagcaggga tatcgtacat ctattgctct 2340 acctcagggg tccagtgata cccactagatcttccaagga aaaacataat tctttcaaac 2400 ggtgtgtatt tggcaaagag ctcttcaaatctgggagagg gacttcctca aggttttcct 2460 gtgtgcagtg gatccacata gctaatatgacagctagtca gttgacaggg accacccaca 2520 gtaagcacca tggtcaggga ggtggcaggaggtgcaaaga cagaagtatt gagagaaaca 2580 ccaagactct agtggaggaa ttaattcaatgggagatagt ataaaataca tagaaaacac 2640 aagtaacaga aacctggttg aaatgcttaactagagtcaa ttagatgtgc aggagtaagt 2700 agtataagaa gaatcaagtc cgagagtgatcaggaaatga gtattaaaca gtatttgaaa 2760 cagagaacgt gtcccagggc ccaaaagtcagaagggcccc accagccagg aaagttgttt 2820 caatgctgta agtaggtgta gccaagggaagccaggacta tctgatatac ggtagcaggg 2880 gtttacggct gccaggggaa aataactcatcaagtgttgg actttcaatt ataagatcga 2940 atttaatttc ctttccctca ttctgcagcaatcagaatac acaatcttaa ccactcggtc 3000 cttagtggtt ttgttccatt ttgcattgggtattttcact gcctcataga gtctatttca 3060 agtgtttggc tgaaagggct ttttgcatttgcatgttctg agttcagatt ctgctggtgc 3120 acccaagcat tatgggaaca ggaactcaacttagctcttc cagtagaggg gtgagggatt 3180 ctgcttttca aattcataac attgatctttttatgcaaga tttccattta cagttgaata 3240 agtacttcat atttttccat cattagacaaatacaaaatg gactaaataa ttttaagaga 3300 tagtggaggc agcagggggt acagacttccttcttagaga gtgtcagaga atatgctccc 3360 aatggtggaa aggaagattt acagtctagcggctaagtac ctcctacaca tttcccatca 3420 atcagaaaat agacaggtac actaaagggacctgagaact cctcttgtaa tttcaacaca 3480 cccaaaatca agggcctgga tgccagcagctgcagcaagc aggtttttcc tccctgttga 3540 gcaagacagg tgaggcaaga taggacttggctttcttaca tgatgcggta acttgtgact 3600 tgagtctttt tccctaattt gctagtgggaagaaaaatag ctgagctttc taaaatgata 3660 gctctctatt tttaaatgaa tttgaaaagtcgattaaatt atgtatttta ttgcctctga 3720 gtatcatatt aaatgaatat tttattttaaaggcttaaat aaatgaaaat gatttttgt 3779 2 2020 DNA Homo sapiens 2aagcttgcta gaaagaaaac tctccctaga cttccagaat cagaatttgc attttaacaa 60gccccccagg ggaatcctgt tcacactgag ttttgagaag ctctgttcta agatattact 120ccaatcccca gcaagccaaa ccgaccactt tcttcacata atcagaacgc tcggttccag 180tatttccaga taccagccat aggaaaacac gttgttctct catttccttt aaccttatct 240aaagccactc aatttctcaa cccttaagtc gtcatctgta aaatgggagt gctaatccta 300cttaatttaa gatgattaag ttgaataata aattagaatt tgtcctacaa attgtgaatc 360atgatcggaa tattcaattt tattattact ctgtgtctaa ctcaataact gggttataaa 420ctctgaaaac aggacaatct cttctctacc tctttcttta tcttccctgt atactgaaat 480acagcgttct gcccagcatg aggtcgacta tttgacattt acaaaaatct ctcttggaat 540ccccctgcca aaaaaaaaaa aatcgaattt caatttatga ctccaagcac aaagagatcc 600tgtcactttg acctaattct ctgaggttca tccctctaaa taattgtatt gtctccatct 660ataaagtact ctgttactat gacaacgcac acataattgc atgtgtaagt agttgagggt 720ggctgtgcaa gtcagactcc tttccaccag gaaacacaga aaacattcta tcttcccaaa 780tgttactctc tcagatttgc accaagaccc tagaagtttc ttaaatttcc atatacttta 840tggacactac aaagcaattc caggaaacac tgatacagta gcaaaactac tgggagtgga 900aattggatgg agatctgggt cccagttcag agtctgacac ctgatttgca acataattag 960gcacagggca ctcaactatt cagggcccta aacccttcta ttgaaaggga aatgggagaa 1020caatatgtgt tcctatggct cagtccctat aagattctgt actattcaga gttgatttta 1080agtgtcactt aactgaaatt atccaacaaa ccttcatggc atgaaacatt aacacagctc 1140tttttatatg gcatggttcc tatggctcaa tccctataag attctgtact agttcagagt 1200tgattttaaa agtcacttaa ctgaaattat ccaacaaacc ctcgaggaca ttaaacatt 1260acgtggctct ttttatatgg catggttcat tatcatgcca ataaatgatt aatcgtaat 1320ctctgtcttg accaataatt ttgctggact tttgtgattc acaacgtgct ctgtgttta 1380atgctacctc ttgaaactga catcctagct ttattgtttt ttattacttc cctaagtgg 1440ctttcaaaag agacaccaag tgacatattt ttaggagggg tttaaaagtt tgatgggta 1500gaagtaaacg ttgcttaact caaccagcag cagagccagg gcccagggac cagccttct 1560gtggacttgg cctttccagc agcagaccca gcaatgaatg atccgaacag ctggtggac 1620aatgcaacag tttgctctgg tgcatcctgt gtggtacctg agagcaattt cataacatc 1680ctaagtgtgg tcctaagtac ggtgctgacc atcctgttgg ccttggtgat gtctccatg 1740ggatgcaacg tggaaatcaa gaaatttcta gggcacataa agcggccgtg ggcatttgt 1800gttggcttcc tctgtcagtt tggaatcatg cccctcacag gattcatcctgtcggtggcc 1860tttgacatcc tcccgctcca ggccgtagtg gtgctcatta taggatgct ccctggagga 1920actgcctcca atatcttggc ctattgggtc gatggcgaca tggacctgg gtaagattat 1980ctatacctct aactactgca atcaccttgg agtaatcctt 2020 3 281 DNA Homo sapiens3 ttggatttgc tttgactcat gattgctggg ttcacttatt cctttttttt tcagattggg 60tccatcgcgg gcgccatcct cattgtgctc atagctgtgg ttggaggaat attgtaccaa 120agcgcctgga tcattgctcc caaactgtgg attataggaa caatatttcc tgtggcgggt 180tactccctgg ggtttcttct ggctagaatt gctggtctac cctggtacag gtatggcatt 240tagtaaatca cattggggat tttaatctgt aatatctgtt g 281 4 348 PRT Homo sapiens4 Met Asn Asp Pro Asn Ser Cys Val Asp Asn Ala Thr Val Cys Ser Gly 1 5 1015 Ala Ser Cys Val Val Pro Glu Ser Asn Phe Asn Asn Ile Leu Ser Val 20 2530 Val Leu Ser Thr Val Leu Thr Ile Leu Leu Ala Leu Val Met Phe Ser 35 4045 Met Gly Cys Asn Val Glu Ile Lys Lys Phe Leu Gly His Ile Lys Arg 50 5560 Pro Trp Gly Ile Cys Val Gly Phe Leu Cys Gln Phe Gly Ile Met Pro 65 7075 80 Leu Thr Gly Phe Ile Leu Ser Val Ala Phe Asp Ile Leu Pro Leu Gln 8590 95 Ala Val Val Val Leu Ile Ile Gly Cys Cys Pro Gly Gly Thr Ala Ser100 105 110 Asn Ile Leu Ala Tyr Trp Val Asp Gly Asp Met Asp Leu Ser ValSer 115 120 125 Met Thr Thr Cys Ser Thr Leu Leu Ala Leu Gly Met Met ProLeu Cys 130 135 140 Leu Leu Ile Tyr Thr Lys Met Trp Val Asp Ser Gly SerIle Val Ile 145 150 155 160 Pro Tyr Asp Asn Ile Gly Thr Ser Leu Val AlaLeu Val Val Pro Val 165 170 175 Ser Ile Gly Met Phe Val Asn His Lys TrpPro Gln Lys Ala Lys Ile 180 185 190 Ile Leu Lys Ile Gly Ser Ile Ala GlyAla Ile Leu Ile Val Leu Ile 195 200 205 Ala Val Val Gly Gly Ile Leu TyrGln Ser Ala Trp Ile Ile Ala Pro 210 215 220 Lys Leu Trp Ile Ile Gly ThrIle Phe Pro Val Ala Gly Tyr Ser Leu 225 230 235 240 Gly Phe Leu Leu AlaArg Ile Ala Gly Leu Pro Trp Tyr Arg Cys Arg 245 250 255 Thr Val Ala PheGlu Thr Gly Met Gln Asn Thr Gln Leu Cys Ser Thr 260 265 270 Ile Val GlnLeu Ser Phe Thr Pro Glu Glu Leu Asn Val Val Phe Thr 275 280 285 Phe ProLeu Ile Tyr Ser Ile Phe Gln Leu Ala Phe Ala Ala Ile Phe 290 295 300 LeuGly Phe Tyr Val Ala Tyr Lys Lys Cys His Gly Lys Asn Lys Ala 305 310 315320 Glu Ile Pro Glu Ser Lys Glu Asn Gly Thr Glu Pro Glu Ser Ser Phe 325330 335 Tyr Lys Ala Asn Gly Gly Phe Gln Pro Asp Glu Lys 340 345

1 A method for the diagnosis of a polymorphism in SLC10A2 in a human,which method comprises determining the sequence of the human at one ormore of the following positions: positions582,664,727,792,890,1073,1103,1384,1466,1484,1545,1646,1683 and 1765 asdefined by the position in SEQ ID NO: 1; position 1982 as defined by theposition in SEQ ID NO: 2; position 258 as defined by the position in SEQID NO: 3; and positions 65, 98, 159, 290, 296 and 316 as defined by theposition in SEQ ID NO:
 4. and determining the status of the human byreference to polymorphism in SLC10A2. 2 The method for diagnosisaccording to claim one which the polymorphism in SLC10A2 is any one ofthe following: Allele in SEQ ID Variant Ref. Sequence Position NO xAllele SEQ ID NO: 1 582 C G SEQ ID NO: 1 664 T C SEQ ID NO: 1 727 C TSEQ ID NO: 1 792 C T SEQ ID NO: 1 890 G A SEQ ID NO: 1 1073 G A SEQ IDNO: 1 1103 C T SEQ ID NO: 1 1384 G T SEQ ID NO: 1 1466 C T SEQ ID NO: 11484 T C SEQ ID NO: 1 1545 G A SEQ ID NO: 1 1646 A T SEQ ID NO: 1 1683 TC SEQ ID NO: 1 1765 T C SEQ ID NO: 2 1982 T C SEQ ID NO: 3 258 G A SEQID NO: 4 65 P L SEQ ID NO: 4 98 V I SEQ ID NO: 4 159 V I SEQ ID NO: 4290 P S SEQ ID NO: 4 296 F L SEQ ID NO: 4 316 G E

3 Use of a diagnostic method as defined in claim 1 to assess thepharmacogenetics of a drug acting at SLC10A2. 4 A polynucleotidecomprising at least 20 bases of the human SLC10A2 gene and comprising anallelic variant selected from any one of the following: Variant Ref.Sequence Position Allele SEQ ID NO: 1 582 G SEQ ID NO: 1 664 C SEQ IDNO: 1 727 T SEQ ID NO: 1 792 T SEQ ID NO: 1 890 A SEQ ID NO: 1 1073 ASEQ ID NO: 1 1103 T SEQ ID NO: 1 1384 T SEQ ID NO: 1 1466 T SEQ ID NO: 11484 C SEQ ID NO: 1 1545 A SEQ ID NO: 1 1646 T SEQ ID NO: 1 1683 C SEQID NO: 1 1765 C SEQ ID NO: 2 1982 C SEQ ID NO: 3 258 A

5 A nucleotide primer which can detect a polymorphism as defined inclaim
 1. 6 An allele specific primer capable of detecting a SLC10A2 genepolymorphism as defined in claim
 1. 7 An allele-specific oligonucleotideprobe capable of detecting a SLC10A2 gene polymorphism as defined inclaim
 1. 8 Use of a SLC10A2 gene polymorphism as defined in claim 1 as agenetic marker in a linkage study. 9 A method of treating a human inneed of treatment with a SLC10A2 drug in which the method comprises: i)diagnosis of a polymorphism in SLC10A2 in the human, which diagnosispreferably comprises determining the sequence at one or more of thefollowing positions: positions582,664,727,792,890,1073,1103,1384,1466,1484,1545,1646,1683 and 1765 asdefined by the position in SEQ ID NO: 1; position 1982 as defined by theposition in SEQ ID NO: 2; position 258 as defined by the position in SEQID NO: 3; and positions 65, 98, 159, 290, 296 and 316 as defined by theposition in SEQ ID NO:
 4. and determining the status of the human byreference to polymorphism in SLC10A2; and ii) administering an effectiveamount of the drug. 10 An allelic variant of human SLC10A2 polypeptidecomprising at least one of the following: Position Allelic variant SEQID NO: 4 65 L SEQ ID NO: 4 98 I SEQ ID NO: 4 159 I SEQ ID NO: 4 290 SSEQ ID NO: 4 296 L SEQ ID NO: 4 316 E

or a fragment thereof comprising at least 10 amino acids provided thatthe fragment comprises at least one allelic variant. 11 An antibodyspecific for an allelic variant of human SLC10A2 polypeptide as definedin claim 10.